Mammary nonmuscle myosin II (NM II) plays an important role in cancer cell migration. LPA is well known as a factor provoking diverse cellular responses including migration, chemotaxis, and invasion. Therefore we examined the role of NM II and its regulation by LPA in cancer cell migration. LPA induces migration by activating the LPA1 receptor which promotes phosphorylation of the 20kDa nonmuscle myosin II light chain through activation of Rho kinase (ROCK). LPA-induced migration and invasion are attenuated by specific inhibitors including the C3 cell-permeable transferase and the ROCK inhibitor, Y-27632. We demonstrate that NM II plays an important role in LPA-induced migration and invasion by inhibiting its cellular function with blebbistatin and shRNA lentivirus directed against the NM II-A or NM II-B heavy chain, to stably silence NM II-A and NM II-B expression. Interestingly, and in contrast to previous findings using MCF 10A cells (Even-Ram et al. Nat. Cell Biol. 2007, 9:299-309), inhibition or loss of either NM II-A (or NM II-B) in the breast cancer cell line 4T1 results in a decrease in migration and invasion. On the other hand our findings are similar to those of Betapudi et al. (Cancer Res. 2006, 66:4725-4733) who found that depletion of NM II-A impaired migration of MDA-MB-231 cells. These cells, which we show are also dependent on LPA signaling for invasion and migration, are also slowed by depletion of NM II-A in our experiments. Thus, these results suggest defined pathways for signaling through the LPA1 receptor to promote LPA-mediated NM II activation and subsequent cell migration in breast cancer cells.